The sirtuin enzymes are NAD+ dependent acetyl-lysine deacetylases. Their catalytic mechanism utilizes NAD+ as a substrate to effect removal of an acetyl group from an acetyl-lysine residue of the protein substrate to form nicotinamide, 2′ O-acetyl-ADP-ribose (AADPR), and the deacetylated substrate (Sauve et al., Curr. Med. Chem., 11: 807-826 (2004)). In mammals, the seven family members of the sirtuin enzymes (SirT1 to SirT7) are responsible for the regulation of a broad range of cellular processes through their vast array of substrates and sub-cellular locations (Michishita et al., Mol. Biol. Cell, 16: 4623-4653 (2005)). SirT1 is localized to the nucleus and is responsible for modulating cellular stress response through the deacetylation of histones and transcription factors (Dali-Youcef et al., Ann. Med., 39: 335 (2007); Motta et al., Cell, 137: 560-570 (2009); Nemoto et al., J. Biol. Chem., 280: 16456 (2005); Picard et al., Nature, 429: 771-776 (2004)). SirT2, localized predominately in the cytosol, has been identified as an α-tubulin deacetylase (North et al., Mol. Cell, 11: 437-444 (2003)), however, the protein has been shown to translocate to the nucleus to regulate the deacetylation of FOXO transcription factors (North et al., supra; Wang et al., Aging Cell, 6: 505-514 (2007); Jing et al., Cell Metab., 6: 105-114 (2007)). SirT3 and SirT5 are mitochondrial deacetylases, the former being shown to deacetylate enzymes involved in energy regulation and metabolism (Schwer et al., Proc. Nat. Acad. Sci. U.S.A., 103: 10224-10229 (2006); Ahn et al., J. Biol. Chem., 282: 33583-33589 (2007)). SirT5 activity is involved in regulation of the urea cycle, although much is still unknown regarding the enzyme's physiological substrates (Nakagawa et al., Cell, 137: 560-570 (2009)). SirT6, localized to the nucleus, is crucial for telomere maintenance through the deacetylation of histone H3, and it also modulates Hif1alpha and NFκB activities.
Protein acetylation has been identified in the nucleus, cytoplasm, and mitochondria. Furthermore, several independent proteomic studies have identified the number of acetylated proteins to be in the thousands, thereby highlighting the potential global role of sirtuin deacetylation in cell maintenance and function (Choudhary et al., Science, 325: 834-840 (2009); Yang et al., Cell, 31: 449-461 (2008); Zhao et al., Science, 327: 1000-1004 (2010)). The most characterized activity catalyzed by sirtuins is the deacetylation of cellular proteins, shown to be a function of SirT1, SirT2, SirT3, SirT5, and SirT6. SirT6 has also been shown to catalyze auto-ADP-ribosyl transfer; however, the enzymatic consequence and physiological relevance of this modification remains unknown (Liszt et al., J. Biol. Chem., 280: 21313-21320 (2005)). By far, the mitochondrial SirT4 and the nucleolar SirT7 are the least characterized of the sirtuin family members. SirT4 has been reported to catalyze ADP-ribosyl transfer to glutamate dehydrogenase and has been implicated in the regulation of insulin secretion; although, the mechanism of catalysis is undetermined (Ahn et al., supra.). SirT7 has been implicated in the regulation of cell proliferation, prevention of cardiomyocyte death, and ribosomal DNA transcription (Vakhrusheva et al., Circ. Res., 102: 703-710 (2008); Vakhrusheva et al., J. Physiol. Pharm., 59: 201-212 (2008); Ford et al., Genes Dev., 20: 1075-1080 (2006)). These findings were observed as consequences of SirT7 knockdown or mutation, and both the enzymatic activity and potential substrates are uncharacterized.
Current methodologies have implicated the involvement of sirtuins in processes that protect against a number of age-related processes and diseases including Type-II diabetes (Imai et al., Trends Pharmacol Sci., 31: 212-220 (2010)) and Alzheimer's disease (Gan et al., Neuron, 58: 10-14 (2008)). However, many of the studies, such as transgenic models of overexpression or enzyme knock-down, are limited to qualitative observations that provide information regarding the global cellular effects of sirtuin activity. There is a demand for the development of a tool that possesses the ability to specifically probe for sirtuin activity in a biologically and pathologically relevant context, rather than using downstream cellular effects as an activity readout.